Assembly of the 20S proteasome

The proteasome is a cellular protease responsible for the selective degradation of the majority of the intracellular proteome. It recognizes, unfolds, and cleaves proteins that are destined for removal, usually by prior attachment to polymers of ubiquitin. This macromolecular machine is composed of two subcomplexes, the 19S regulatory particle (RP) and the 20S core particle (CP), which together contain at least 33 different and precisely positioned subunits. How these subunits assemble into functional complexes is an area of active exploration. Here we describe the current status of studies on the assembly of the 20S proteasome (CP). The 28-subunit CP is found in all three domains of life and its cylindrical stack of four heptameric rings is well conserved. Though several CP subunits possess self-assembly properties, a consistent theme in recent years has been the need for dedicated assembly chaperones that promote on-pathway assembly. To date, a minimum of three accessory factors have been implicated in aiding the construction of the 20S proteasome. These chaperones interact with different assembling proteasomal precursors and usher subunits into specific slots in the growing structure. This review will focus largely on chaperone-dependent CP assembly and its regulation.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.     

26S蛋白酶体组装的分子机制研究

26S蛋白酶体广泛分布于真核细胞中的胞质和胞核,主要是由20S核心复合物(coreparticle,CP)和19S调节复合物(regulatory particle,RP)组成,它负责细胞大多数蛋白质的降解,在几乎所有生命活动中具有关键的调控作用。26S蛋白酶体的组装是一个非常复杂且高度条理的过程,不同的分子伴侣,如PAC1-4、Ump1、p27、p28和s5b等,参与其中发挥识别及调节作用,以确保高效准确地完成蛋白酶体的组装。本文系统总结分析了20S核心复合物和19S调节复合物的组装过程及调控机制的最近研究进展。     

N-Terminal α7 Deletion of the Proteasome 20S Core Particle Substitutes for Yeast PI31 Function

The proteasome core particle (CP) is a conserved protease complex that is formed by the stacking of two outer α-rings and two inner β-rings. The α-ring is a heteroheptameric ring of subunits α1 to α7 and acts as a gate that restricts entry of substrate proteins into the catalytic cavity formed by the two abutting β-rings. The 31-kDa proteasome inhibitor (PI31) was originally identified as a protein that binds to the CP and inhibits CP activity in vitro, but accumulating evidence indicates that PI31 is required for physiological proteasome activity. To clarify the in vivo role of PI31, we examined the Saccharomyces cerevisiae PI31 ortholog Fub1. Fub1 was essential in a situation where the CP assembly chaperone Pba4 was deleted. The lethality of Δfub1 Δpba4 was suppressed by deletion of the N terminus of α7 (α7ΔN), which led to the partial activation of the CP. However, deletion of the N terminus of α3, which activates the CP more efficiently than α7ΔN by gate opening, did not suppress Δfub1 Δpba4 lethality. These results suggest that the α7 N terminus has a role in CP activation different from that of the α3 N terminus and that the role of Fub1 antagonizes a specific function of the α7 N terminus.     

Characterization of the 20S proteasome of the lepidopteran,Spodoptera frugiperda

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.     

Proteasomes of the yeastS. cerevisiae: genes,structure and functions

Proteasomes are large multicatalytic protease complexes which fulfil central functions in major intracellular proteolytic pathways of the eukaryotic cell. 20S proteasomes are 700 kDa cylindrically shaped particles, found in the cytoplasm and the nucleus of all eukaryotes. They are composed of a pool of 14 different subunits (MW 22–25 kDa) arranged in a stack of 4 rings with 7-fold symmetry. In the yeastSaccharomyces cerevisiae a complete set of 14 genes coding for 20S proteasome subunits have been cloned and sequenced. 26S proteasomes are even larger proteinase complexes (about 1700 kDa) which degrade ubiquitinylated proteins in an ATP-dependent fashionin vitro. The 26S proteasome is build up from the 20S proteasome as core particle and two additional 19S complexes at both ends of the 20S cylinder. Recently existence of a 26S proteasome in yeast has been demonstrated. Several 26S proteasome specific genes have been cloned and sequenced. They share similarity with a novel defined family of ATPases. 20S and 26S proteasomes are essential for functioning of the eukaryotic cell. Chromosomal deletion of 20S and 26S proteasomal genes in the yeastS. cerevisiae caused lethality of the cell. Thein vivo functions of proteasomes in major proteolytic pathways have been demonstrated by the use of 20S and 26S proteasomal mutants. Proteasomes are needed for stress dependent and ubiquitin mediated proteolysis. They are involved in the degradation of short-lived and regulatory proteins. Proteasomes are important for cell differentiation and adaptation to environmental changes. Proteasomes have also been shown to function in the control of the cell cycle.     

An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.     

Substrate access and processing by the 20S proteasome core particle

Intracellular proteolysis is an essential process. In eukaryotes, most proteins in the cytosol and nucleus are degraded by the ubiquitin (Ub)-proteasome pathway. A major component within this system is the 26S proteasome, a 2.5MDa molecular machine, built from more than 31 different subunits. This complex is formed by a cylinder-shaped multimeric complex referred to as the proteolytic 20S proteasome (core particle, CP) capped at each end by another multimeric component called the 19S complex (regulatory particle, RP) or PA700. Structure, assembly and enzymatic mechanism have been elucidated only for the CP, whereas the organization of the RP is less well understood. The CP is composed of 28 subunits, which are arranged as an alpha7beta7beta7alpha7-complex in four stacked rings. The interior of the free core particle, which harbors the active sites, is inaccessible for folded and unfolded substrates and represents a latent state. This inhibition is relieved upon binding of the RP to the CP by formation of the 26S proteasome holoenzyme. This review summarizes the current knowledge of the structural features of 20S proteasomes.     

Dissecting protein‐protein interactions in proteasome assembly: Implication to its self‐assembly

The 26S proteasome is a multi‐catalytic ATP‐dependent protease complex that recognizes and cleaves damaged or misfolded proteins to maintain cellular homeostasis. The 26S subunit consists of 20S core and 19S regulatory particles. 20S core particle consists of a stack of heptameric alpha and beta subunits. To elucidate the structure‐function relationship, we have dissected protein‐protein interfaces of 20S core particle and analyzed structural and physiochemical properties of intra‐alpha, intra‐beta, inter‐beta, and alpha‐beta interfaces. Furthermore, we have studied the evolutionary conservation of 20S core particle. We find the size of intra‐alpha interfaces is significantly larger and is more hydrophobic compared with other interfaces. Inter‐beta interfaces are well packed, more polar, and have higher salt‐bridge density than other interfaces. In proteasome assembly, residues in beta subunits are better conserved than alpha subunits, while multi‐interface residues are the most conserved. Among all the residues at the interfaces of both alpha and beta subunits, Gly is highly conserved. The largest size of intra‐alpha interfaces complies with the hypothesis that large interfaces form first during the 20S assembly. The tight packing of inter‐beta interfaces makes the core particle impenetrable from outer wall of the cylinder. Comparing the three domains, eukaryotes have large and well‐packed interfaces followed by archaea and bacteria. Our findings provide a structural basis of assembly of 20S core particle in all the three domains of life.     

Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails

The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.     

Proteasome disassembly and downregulation is correlated with viability during stationary phase

During prolonged starvation, yeast cells enter a stationary phase (SP) during which the synthesis of many proteins is dramatically decreased. We show that a parallel decrease in proteasome-dependent proteolysis also occurs. The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle (CP) and 19S regulatory particle (RP) components. Proteasomes are reassembled, and proteolysis resumes prior to cell cycle reentry. Free 20S CPs are found in an autoinhibited state in which the N-terminal tails from neighboring alpha subunits are anchored by an intricate lattice of interactions blocking the channel that leads into the 20S CPs. By deleting channel gating residues of CP alpha subunits, we generated an "open channel" proteasome that exhibits faster rates of protein degradation both in vivo and in vitro, indicating that gating contributes to regulation of proteasome activity. This open channel mutant is delayed in outgrowth from SP and cannot survive following prolonged starvation. In summary, we have found that the ubiquitin-proteasome pathway can be subjected to global downregulation, that the proteasome is a target of this regulation, and that proteasome downregulation is linked to survival of SP cells. Maintaining high viability during SP is essential for evolutionary fitness, which may explain the extreme conservation of channel gating residues in eukaryotic proteasomes.     

1H, 13C and 15N resonance assignments of the VWA domain of Saccharomyces cerevisiae Rpn10, a regulatory subunit of 26S proteasome

Rpn10 is a ubiquitin receptor of the 26S proteasome, and plays an important role in poly-ubiquitinated proteins recognition in the ubiquitin–proteasome protein degradation pathway. It is located in the 19S regulatory particle and interacts with several subunits of both lid and base complexes. Bioinformatics analysis of yeast Rpn10 suggests that it contains a von Willebrand (VWA domain) and a C-terminal tail containing a Ub-interacting motif. Studies of Saccharomyces cerevisiae Rpn10 suggested that its VWA domain might participate in interactions with subunit from both lid and base subcomplexes of the 19S regulatory particle. Herein, we report the chemical shift assignments of ¹H, ¹³C and ¹⁵N atoms of the VWA domain of S. cerevisiae Rpn10, which provide the basis for further structural and functional studies of Rpn10 by solution NMR technique.     

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.     

Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.     

Structure of the human 26S proteasome: subunit radial displacements open the gate into the proteolytic core

The 26S proteasome plays an essential role in regulating many cellular processes by the degradation of proteins targeted for breakdown by ubiquitin conjugation. The 26S complex is formed from the 20S core, which contains the proteolytic active sites, and 19S regulatory complexes, which bind to the 20S core to activate it and confer specificity for ubiquitinated protein substrates. We have determined the structure of the human 26S proteasome by electron microscopy and single particle analysis. In our reconstructions the crystallographic structure of each of the subunits of the 20S core can be unambiguously docked by direct recognition of each of their densities. Our results show for the first time that binding of the 19S regulatory particle results in the radial displacement of the adjacent subunits of the 20S core leading to opening of a wide channel into the proteolytic chamber. The analysis of a proteasome complex formed from one 20S core with a single 19S regulatory particle attached serve as control to our observations. We suggest locations for some of the 19S regulatory particle subunits.     

C termini of proteasomal ATPases play nonequivalent roles in cellular assembly of mammalian 26 S proteasome

The 26 S proteasome comprises two multisubunit subcomplexes as follows: 20 S proteasome and PA700/19 S regulatory particle. The cellular mechanisms by which these subcomplexes assemble into 26 S proteasome and the molecular determinants that govern the assembly process are poorly defined. Here, we demonstrate the nonequivalent roles of the C termini of six AAA subunits (Rpt1-Rpt6) of PA700 in 26 S proteasome assembly in mammalian cells. The C-terminal HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) of each of two subunits, Rpt3 and Rpt5, but not that of a third subunit Rpt2, was essential for assembly of 26 S proteasome. The C termini of none of the three non-HbYX motif Rpt subunits were essential for cellular 26 S proteasome assembly, although deletion of the last three residues of Rpt6 destabilized the 20 S-PA700 interaction. Rpt subunits defective for assembly into 26 S proteasome due to C-terminal truncations were incorporated into intact PA700. Moreover, intact PA700 accumulated as an isolated subcomplex when cellular 20 S proteasome content was reduced by RNAi. These results indicate that 20 S proteasome is not an obligatory template for assembly of PA700. Collectively, these results identify specific structural elements of two Rpt subunits required for 26 S proteasome assembly, demonstrate that PA700 can be assembled independently of the 20 S proteasome, and suggest that intact PA700 is a direct intermediate in the cellular pathway of 26 S proteasome assembly.     

Structure and functional analyses of the 26S proteasome subunits from plants – Plant 26S proteasome

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10 strains which grow normally, Physcomitrella rpn10 strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.